US6083925A - Nucleic acid respiratory syncytial virus vaccines - Google Patents

Nucleic acid respiratory syncytial virus vaccines Download PDF

Info

Publication number
US6083925A
US6083925A US09/262,927 US26292799A US6083925A US 6083925 A US6083925 A US 6083925A US 26292799 A US26292799 A US 26292799A US 6083925 A US6083925 A US 6083925A
Authority
US
United States
Prior art keywords
rsv
protein
sequence
nucleotide sequence
host
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US09/262,927
Other languages
English (en)
Inventor
Xiaomao Li
Mary E. Ewasyshyn
Suryaprakash Sambhara
Michel H. Klein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi Pasteur Ltd
Original Assignee
Connaught Laboratories Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/476,397 external-priority patent/US6019980A/en
Priority claimed from US08/896,500 external-priority patent/US6017897A/en
Priority to US09/262,927 priority Critical patent/US6083925A/en
Application filed by Connaught Laboratories Ltd filed Critical Connaught Laboratories Ltd
Assigned to CONNAUGHT LABORATORIES LIMITED reassignment CONNAUGHT LABORATORIES LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: EWASYSHYN, MARY E., LI, XIAOMAO, SAMBHARA, SURYAPRAKASH, KLEIN, MICHEL H.
Priority to MXPA01008966A priority patent/MXPA01008966A/es
Priority to PCT/CA2000/000227 priority patent/WO2000053767A2/en
Priority to JP2000603388A priority patent/JP3602448B2/ja
Priority to NZ514528A priority patent/NZ514528A/xx
Priority to AU29002/00A priority patent/AU778144B2/en
Priority to CA002363593A priority patent/CA2363593A1/en
Priority to US09/570,383 priority patent/US6486135B1/en
Publication of US6083925A publication Critical patent/US6083925A/en
Application granted granted Critical
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

  • the present invention is related to the field of Respiratory Syncytial Virus (RSV) vaccines and is particularly concerned with vaccines comprising nucleic acid sequences encoding the fusion (F) protein of RSV.
  • RSV Respiratory Syncytial Virus
  • Respiratory syncytial virus a negative-strand RNA virus belonging to the Paramyxoviridae family of viruses, is the major viral pathogen responsible for bronchiolitis and pneumonia in infants and young children (ref. 1--Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure).
  • Acute respiratory tract infections caused by RSV result in approximately 90,000 hospitalizations and 4,500 deaths per year in the United States (ref. 2). Medical care costs due to RSV infection are greater than $340 M annually in the United States alone (ref. 3).
  • the main approaches for developing an RSV vaccine have included inactivated virus, live-attenuated viruses and subunit vaccines.
  • the F protein of RSV is considered to be one of the most important protective antigens of the virus. There is a significant similarity (89% identity) in the amino acid sequences of the F proteins from RSV subgroups A and B (ref. 3) and anti-F antibodies can cross-neutralize viruses of both subgroups as well as protect immunized animals against infection with viruses from both subgroups (ref. 4). Furthermore, the F protein has been identified as a major target for RSV-specific cytotoxic T-lymphocytes in mice and humans (ref. 3 and ref. 5).
  • RSV proteins as vaccines may have obstacles.
  • Parenterally administered vaccine candidates have so far proven to be poorly immunogenic with regard to the induction of neutralizing antibodies in seronegative humans or chimpanzees.
  • the serum antibody response induced by these antigens may be further diminished in the presence of passively acquired antibodies, such as the transplacentally acquired maternal antibodies which most young infants possess.
  • a subunit vaccine candidate for RSV consisting of purified fusion glycoprotein from RSV infected cell cultures and purified by immunoaffinity or ion-exchange chromatography has been described (ref. 6).
  • the immune response to immunization with RSV F protein or a synthetic RSV FG fusion protein resulted in a disease enhancement in rodents resembling that induced by a formalin-inactivated RSV vaccine.
  • the association of immunization with disease enhancement using non-replicating antigens suggests caution in their use as vaccines in seronegative humans.
  • Live attenuated vaccines against disease caused by RSV may be promising for two main reasons. Firstly, infection by a live vaccine virus induces a balanced immune response comprising mucosal and serum antibodies and cytotoxic T-lymphocytes. Secondly, infection of infants with live attenuated vaccine candidates or naturally acquired wild-type virus is not associated with enhanced disease upon subsequent natural reinfection. It will be challenging to produce live attenuated vaccines that are immunogenic for younger infants who possess maternal virus-neutralizing antibodies and yet are attenuated for seronegative infants greater than or equal to 6 months of age. Attenuated live virus vaccines also have the risks of residual virulence and genetic instability.
  • DNA encoding a viral antigen can be introduced in the presence of antibody to the virus itself, without loss of potency due to neutralization of virus by the antibodies.
  • the antigen expressed in vivo should exhibit a native conformation and, therefore, should induce an antibody response similar to that induced by the antigen present in the wild-type virus infection.
  • some processes used in purification of proteins can induce conformational changes which may result in the loss of immunogenicity of protective epitopes and possibly immunopotentiation.
  • the expression of proteins from injected plasmid DNAs can be detected in vivo for a considerably longer period of time than that in virus-infected cells, and this has the theoretical advantage of prolonged cytotoxic T-cell induction and enhanced antibody responses.
  • in vivo expression of antigen may provide protection without the need for an extrinsic adjuvant.
  • the ability to immunize against disease caused by RSV by administration of a DNA molecule encoding an RSV F protein was unknown before the present invention.
  • the efficacy of immunization against RSV induced disease using a gene encoding a secreted form of the RSV F protein was unknown. Infection with RSV leads to serious disease.
  • the present invention relates to a method of immunizing a host against disease caused by respiratory syncytial virus, to nucleic acid molecules used therein, and to diagnostic procedures utilizing the nucleic acid molecules.
  • the present invention is directed towards the provision of nucleic acid respiratory syncytial virus vaccines.
  • an immunogenic composition for in vivo administration to a host for the generation in the host of a protective immune response to RSV F protein comprising a non-replicating vector comprising:
  • CTLs cytotoxic T-lymphocytes
  • a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV F protein
  • a second nucleotide sequence located adjacent the first nucleotide sequence to enhance the immunoprotective ability of the RSV F protein when expressed in vivo from the vector in a host;
  • the first nucleotide sequence may be that which encodes a full-length RSV F protein, as seen in FIG. 2 (SEQ ID No: 2).
  • the first nucleotide sequence may be that which encodes an RSV F protein from which the transmembrane region is absent.
  • the latter embodiment may be provided by a nucleotide sequence which encodes a full-length RSV F protein but contains a translational stop codon immediately upstream of the start of the transmembrane coding region, thereby preventing expression of a transmembrane region of the RSV F protein, as seen in FIG. 3 (SEQ. ID No. 4). The lack of expression of the transmembrane region results in a secreted form of the RSV F protein.
  • the first nucleotide sequence may encode a RSV F protein fragment lacking an autologous RSV F signal peptide sequence and may include, in its place, a sequence encoding a heterologous signal peptide sequence which enhances the level of expression of the RSV F protein.
  • One signal peptide which has been found useful in this regard is the signal peptide of Herpes Simplex Virus I (HSV I)gD. Such enhanced expression levels also lead to improve immunogenicity of the vector at the same dosage level.
  • the first nucleotide sequence may also encode a RSV F protein fragment lacking a transmembrane coding region.
  • the second nucleotide sequence may comprise a pair of splice sites to prevent aberrant mRNA splicing, whereby substantially all transcribed mRNA encodes the RSV protein.
  • Such second nucleotide sequence may be located between the first nucleotide sequence and the promoter sequence.
  • Such second nucleotide sequence may be that of rabbit ⁇ -globin intron II, as shown in FIG. 8 (SEQ ID No: 5).
  • a vector encoding the F protein and provided by this aspect of the invention may specifically be pXL2 or pXL4 or p82M35B, as seen in FIGS. 5, 7 or 10, respectively.
  • the promoter sequence may be an immediate early cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • a method of immunizing a host against disease caused by infection with respiratory syncytial virus which comprises administering to the host an effective amount a of non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein or a RSV F protein fragment that generates antibodies and/or CTLs that specifically react with RSV F protein and a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV F protein in the host, which may be a human.
  • the promoter may be an immediate early cytomegalovirus promoter.
  • the nucleotide sequence may encode a truncated RSV F protein lacking the transmembrane region may be that as described above and/or possess a heterologous signal peptide encoding sequence.
  • the vector may contain a second nucleotide sequence located adjacent a first nucleotide sequence and effective to enhance the immunoprotective ability of the RSV F protein expressed by the first nucleotide sequence may be used to immunize a host.
  • Specific non-replicating vectors which may be used in this aspect of the invention are those identified as plasmid vectors pXL2, pXL4 and p82M35B in FIGS. 5, 7 and 10 respectively.
  • the present invention also includes a novel method of using a gene encoding an RSV F protein or a RSV F protein fragment that generates antibodies and/or CTLs that specifically react with RSV F protein to protect a host against disease caused by infection with respiratory syncytial virus, which comprises:
  • control sequence operatively linking the gene to at least one control sequence to produce a non-replicating vector, said control sequence directing expression of the RSV F protein when said vector is introduced into a host to produce an immune response to the RSV F protein or fragment thereof, and
  • the procedure provided in accordance with this aspect of the invention may further include the step of:
  • the present invention includes a method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus, which comprises:
  • control sequence operatively linking the first nucleotide sequence to at least one control sequence to produce a non-replicating vector, the control sequence directing expression of the RSV F protein when introduced into a host to produce an immune response to the RSV F protein when expressed in vivo from the vector in a host, and
  • the first nucleotide sequence further may be operatively linked to a second nucleotide sequence to enhance the immunoprotective ability of the RSV F protein when expressed in vivo from the vector in a host.
  • the vector may be a plasmid vector selected from pXL2, pXL4 and p82M35B.
  • the invention further includes a vaccine for administration to a host, including a human host, produced by this method as well as immunogenic compositions comprising an immunoeffective amount of the vectors described herein.
  • CTLs cytotoxic T-lymphocytes
  • the non-replicating vector employed to elicit the antibodies may be a plasmid vector which is pXL1, pXL2, pXL3, pXL4 or p82M35B.
  • the invention also includes a diagnostic kit for detecting the presence of an RSV F protein in a sample, comprising:
  • a non-replicating vector comprising a first nucleotide sequence encoding an RSV F protein or a RSV F protein fragment that generates antibodies that specifically react with RSV F protein and a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV F protein in a host immunized therewith to produce antibodies specific for the RSV F protein;
  • isolation means to isolate said RSV F protein specific antibodies
  • the present invention is further directed to a method for producing RSV F protein specific polyclonal antibodies comprising the use of the immunization method described herein, and further comprising the step of isolating the RSV F protein specific polyclonal antibodies from the immunized animal.
  • the present invention is also directed to a method for producing monoclonal antibodies specific for an F protein of RSV, comprising the steps of:
  • a second nucleotide sequence located adjacent said first nucleotide sequence to enhance the immunoprotective ability of said RSV F protein when expressed in vivo from said vector in a host.
  • RSV F protein is used to define (1) a full-length RSV F protein, such proteins having variations in their amino acid sequences including those naturally occurring in various strains of RSV, (2) a secreted form of RSV F protein lacking a transmembrane region, and (3) functional analogs of the RSV F protein.
  • a first protein is a "functional analog" of a second protein if the first protein is immunologically related to and/or has the same function as the second protein.
  • the functional analog may be, for example, a fragment of the protein or a substitution, addition or deletion mutant thereof. Included are RSV F protein fragments that generate antibodies and/or CTLs that specifically react with RSV F protein.
  • FIG. 1 illustrates a restriction map of the gene encoding the F protein of Respiratory Syncytial Virus
  • FIGS. 2A, 2B, 2C, 2D and 2E show the nucleotide sequence of the gene encoding the membrane attached form of the F protein of Respiratory Syncytial Virus (SEQ ID No: 1) as well as the amino acid sequence of the RSV F protein encoded thereby (SEQ ID No: 2);
  • FIGS. 3A, 3B, 3C and 3D show the nucleotide sequence of the gene encoding the secreted form of the RSV F protein lacking the transmembrane region (SEQ ID No: 3) as well as the amino acid sequence of the truncated RSV F protein lacking the transmembrane region encoded thereby (SEQ ID No: 4);
  • FIGS. 4A, 4B, 4C and 4D show the construction of plasmid pXL1 containing the gene encoding a secreted form of the RSV F protein lacking the transmembrane region;
  • FIGS. 5A, 5B, 5C and 5D show the construction of plasmid pXL2 containing a gene encoding a secreted form of the RSV F protein lacking the transmembrane region and containing the rabbit ⁇ -globin Intron II sequence;
  • FIGS. 6A, 6B, 6C and 6D show the construction of plasmid pXL3 containing the gene encoding a full length membrane attached form of the RSV F protein;
  • FIGS 7A, 7B, 7C and 7D show the construction of plasmid pXL4 containing a gene encoding a membrane attached form of the RSV F protein and containing the rabbit ⁇ -globin Intron II sequence;
  • FIG. 8 shows the nucleotide sequence for the rabbit ⁇ -globin Intron II sequence (SEQ ID No. 5);
  • FIG. 9 shows the lung cytokine expression profile in DNA-immunized mice after RSV challenge
  • FIG. 10 is a schematic showing the assembly of plasmid p82M35B containing a gene encoding a secreted form of the RSV F protein lacking the transmembrane region, the rabbit ⁇ -globin Intron II sequence and the signal peptide sequence HSV I gD;
  • FIG. 11 shows the nucleotide sequence of plasmid VR-1012 (SEQ ID No: 6).
  • FIG. 12 shows DNA (SEQ ID No: 7) and derived amino acid (SEQ ID No: 8) sequence of the HSV gD signal peptide sequence, synthesized as a synthetic oligopeptide.
  • the present invention relates generally to polynucleotide, including DNA, immunization to obtain protection against infection by respiratory syncytial virus (RSV) and to diagnostic procedures using particular vectors.
  • RSV respiratory syncytial virus
  • several recombinant vectors were constructed to contain a nucleotide sequence encoding an RSV F protein.
  • the nucleotide sequence of the full length RSV F gene is shown in FIG. 2 (SEQ ID No: 1). Certain constructs provided herein include the nucleotide sequence encoding the full-length RSV F (SEQ ID No: 2) protein while others include an RSV F gene modified by insertion of termination codons immediately upstream of the transmembrane coding region (see FIG. 3, SEQ ID No: 3), to prevent expression of the transmembrane portion of the protein and to produce a secreted or truncated RSV F protein lacking a transmembrane region (SEQ ID No. 4).
  • constructs provided herein include a nucleic acid sequence encoding a heterologous signal peptide sequence rather than the native signal peptide sequence to provide for enhanced protein expression and increased immunogenicity.
  • the signal peptide sequence for HSV I gD is employed.
  • other heterologous signal peptides may be employed, such as that of human tissue plasminogen activator (TPA).
  • the nucleotide sequence encoding the RSV F protein is operatively coupled to a promoter sequence for expression of the encoded RSV F protein.
  • the promoter sequence may be the immediately early cytomegalovirus (CMV) promoter. This promoter is described in ref. 13. Any other convenient promoter may be used, including constitutive promoters, such as, Rous Sarcoma Virus LTRs, and inducible promoters, such as metallothionine promoter, and tissue specific promoters.
  • the vectors provided herein when administered to an animal, effect in vivo RSV F protein expression, as demonstrated by an antibody response in the animal to which it is administered.
  • Such antibodies may be used herein in the detection of RSV protein in a sample, as described in more detail below.
  • the encoded RSV F protein is in the form of an RSV F protein from which the transmembrane region is absent, such as plasmid pXL1 (FIG. 4)
  • the administration of the vector conferred protection in mice and cotton rats to challenge by live RSV virus neutralizing antibody and cell mediated immune responses and an absence of immunopotentiation in immunized animals, as seen from the Examples below.
  • the recombinant vector also may include a second nucleotide sequence located adjacent the RSV F protein encoding nucleotide sequence to enhance the immunoprotective ability of the RSV F protein when expressed in vivo in a host. Such enhancement may be provided by increased in vivo expression, for example, by increased mRNA stability, enhanced transcription and/or translation.
  • This additional sequence preferably is located between the promoter sequence and the RSV F protein-encoding sequence.
  • This enhancement sequence may comprise a pair of splice sites to prevent aberrant mRNA splicing during transcription and translation so that substantially all transcribed mRNA encodes an RSV F protein.
  • the rabbit ⁇ -globin Intron II sequence shown in FIG. 7 may provide such splice sites, as also described in ref. 15.
  • the construct containing the Intron II sequence, CMV promoter and nucleotide sequence coding for the full-length RSV F protein, i.e. plasmid pXL4 (FIG. 7) also conferred protection in mice to challenge with live RSV, as seen from the Examples below.
  • the vector provided herein may also comprise a third nucleotide sequence encoding a further antigen from RSV, an antigen from at least one other pathogen or at least one immunomodulating agent, such as cytokine.
  • a third nucleotide sequence encoding a further antigen from RSV, an antigen from at least one other pathogen or at least one immunomodulating agent, such as cytokine.
  • Such vector may contain said third nucleotide sequence in a chimeric or a bicistronic structure.
  • vectors containing the third nucleotide sequence may be separately constructed and coadministered to a host, with the nucleic acid molecule provided herein.
  • Immunogenic compositions suitable to be used as vaccines, may be prepared from the RSV F genes and vectors as disclosed herein.
  • the vaccine elicits an immune response in a subject which includes the production of anti-F antibodies.
  • Immunogenic compositions, including vaccines, containing the nucleic acid may be prepared as injectables, in physiologically-acceptable liquid solutions or emulsions for polynucleotide administration.
  • the nucleic acid may be associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a nucleic acid liposome (for example, as described in WO 9324640, ref. 17) or the nucleic acid may be associated with an adjuvant, as described in more detail below.
  • Liposomes comprising cationic lipids interact spontaneously and rapidly with polyanions such as DNA and RNA, resulting in liposome/nucleic acid complexes that capture up to 100% of the polynucleotide.
  • polyanions such as DNA and RNA
  • the polycationic complexes fuse with cell membranes, resulting in an intracellular delivery of polynucleotide that bypasses the degradative enzymes of the lysosomal compartment.
  • Published PCT application WO 94/27435 describes compositions for genetic immunization comprising cationic lipids and polynucleotides.
  • Agents which assist in the cellular uptake of nucleic acid such as calcium ions, viral proteins and other transfection facilitating agents, may advantageously be used.
  • Polynucleotide immunogenic preparations may also be formulated as microcapsules, including biodegradable time-release particles.
  • U.S. Pat. No. 5,151,264 describes a particulate carrier of a phospholipid/glycolipid/polysaccharide nature that has been termed Bio Vendels Supra Mole vides (BVSM).
  • BVSM Bio Vendels Supra Mole vides
  • U.S. Pat. No. 5,075,109 describes encapsulation of the antigens trinitrophenylated keyhole limpet hemocyanin and staphylococcal enterotoxin B in 50:50 poly (DL-lactideco-glycolide).
  • Other polymers for encapsulation are suggested, such as poly(glycolide), poly(DL-lactide-co-glycolide), copolyoxalates, polycaprolactone, poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters and poly(8-hydroxybutyric acid), and polyanhydrides.
  • WO 91/06282 describes a delivery vehicle comprising a plurality of bioadhesive microspheres and antigens.
  • the microspheres being of starch, gelatin, dextran, collagen or albumin.
  • This delivery vehicle is particularly intended for the uptake of vaccine across the nasal mucosa.
  • the delivery vehicle may additionally contain an absorption enhancer.
  • the RSV F genes and vectors may be mixed with pharmaceutically acceptable excipients which are compatible therewith.
  • excipients may include, water, saline, dextrose, glycerol, ethanol, and combinations thereof.
  • the immunogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.
  • Immunogenic compositions and vaccines may be administered parenterally, by injection subcutaneously, intravenously, intradermally or intramuscularly, possibly following pretreatment of the injection site with a local anesthetic.
  • the immunogenic compositions formed according to the present invention may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces.
  • the immunogenic composition may be administered to mucosal surfaces by, for example, the nasal or oral (intragastric) routes.
  • binders and carriers may include, for example, polyalkalene glycols or triglycerides.
  • Oral formulations may include normally employed incipients, such as, for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate.
  • the immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize the PSV F protein and antibodies thereto, and if needed, to produce a cell-mediated immune response.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of about 1 ⁇ g to about 11 mg of the RSV F genes and vectors. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations.
  • the dosage may also depend on the route of administration and will vary according to the size of the host.
  • a vaccine which protects against only one pathogen is a monovalent vaccine.
  • Vaccines which contain antigenic material of several pathogens are combined vaccines and also belong to the present invention. Such combined vaccines contain, for example, material from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens.
  • Immunogenicity can be significantly improved if the vectors are co-administered with adjuvants, commonly used as 0.05 to 0.1 percent solution in phosphate-buffered saline.
  • adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves.
  • Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system.
  • Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.
  • Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Thus, adjuvants have been identified that enhance the immune response to antigens. Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.
  • extrinsic adjuvants and other immunomodulating material can provoke potent immune responses to antigens.
  • these include saponins complexed to membrane protein antigens to produce immune stimulating complexes (ISCOMS), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as monophoryl lipid A, QS 21 and polyphosphazene.
  • ISCOMS immune stimulating complexes
  • MDP muramyl dipeptide
  • LPS lipopolysaccharide
  • the vector comprising a first nucleotide sequence encoding an F protein of RSV may be delivered in conjunction with a targeting molecule to target the vector to selected cells including cells of the immune system.
  • the polynucleotide may be delivered to the host by a variety of procedures, for example, Tang et al. (ref. 10) disclosed that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice, while Furth et al. (ref. 11) showed that a jet injector could be used to transfect skin, muscle, fat and mammary tissues of living animals.
  • Tang et al. disclosed that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice
  • Furth et al. showed that a jet injector could be used to transfect skin, muscle, fat and mammary tissues of living animals.
  • the RSV F genes and vectors of the present invention are useful as immunogens for the generation of anti-F antibodies for use in immunoassays, including enzyme-linked immunosorbent assays (ELISA), RIAs and other non-enzyme linked antibody binding assays or procedures known in the art.
  • ELISA assays the vector first is administered to a host to generate antibodies specific to the RSV F protein.
  • RSV F-specific antibodies are immobilized onto a selected surface, for example, a surface capable of binding the antibodies, such as the wells of a polystyrene microtiter plate.
  • a nonspecific protein such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard to the test sample may be bound to the selected surface.
  • BSA bovine serum albumin
  • the immobilizing surface is then contacted with a sample, such as clinical or biological materials, to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • This procedure may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween.
  • BGG bovine gamma globulin
  • PBS phosphate buffered saline
  • the sample is then allowed to incubate for from about 12 to 4 hours, at temperatures such as of the order of about 120° to 37° C.
  • the sample-contacted surface is washed to remove non-immunocomplexed material.
  • the washing procedure may include washing with a solution, such as PBS/Tween or a borate buffer. Following formation of specific immunocomplexes between the test sample and the bound RSV F specific antibodies, and subsequent washing, the occurrence, and even amount
  • This Example describes the construction of vectors containing the RSV F gene.
  • FIG. 1 shows a restriction map of the gene encoding the F protein of Respiratory Syncytial Virus
  • FIG. 2 shows the nucleotide sequence of the gene encoding the full-length RSV F protein (SEQ ID No: 1) and the deduced amino acid sequence (SEQ ID No: 2).
  • FIG. 3 shows the gene encoding the secreted RSV F protein (SEQ ID No: 3) and the deduced amino acid sequence (SEQ ID No: 4).
  • a set of four plasmid DNA constructs were made (as shown schematically in FIGS. 4 to 7) in which cDNA encoding the RSV-F was subcloned downstream of the immediate-early promoter, enhancer and intron A sequences of human cytomegalovirus (CMV) and upstream of the bovine growth hormone (BGH) poly-A site.
  • CMV cytomegalovirus
  • BGH bovine growth hormone
  • the 1.6 Kb Sspl-PstI fragment containing the promoter, enhancer and intron A sequences of CMV Towne strain were initially derived from plasmid pRL43a obtained from Dr. G. S. Hayward of Johns Hopkins University (ref.
  • Either the 1.6 kb EcoRI-BamHI fragment or the 2.2 kb ClaI-BamHI fragment was then excised from the pSG5 construct, filled-in with Klenow and subcloned at the SmaI site of the pBluescript II SK +/- construct containing the promoter and intron A sequences.
  • the 0.6 kb ClaI-EcoPI fragment derived from pSG5 contained the intron II sequences from rabbit ⁇ -globin.
  • the plasmids were digested with HindIII, filled-in with Klenow, and digested with XbaI to yield either a 3.2 or a 3.8 Kb fragment.
  • the full length RSV F cDNA was excised as a 1.9 kb EcoRI fragment from a recombinant pBluescript M13-SK (Stratagene) containing the insert (ref. 18 and WO 93/14207) and subcloned at the EcoRI site of pSG5 (Stratagene).
  • Plasmids pXL1 and pXL2 were made to express a truncated/secretory form of the F protein which carried stop codons resulting in a C-terminal deletion of 48 amino acids including the transmembrane (TM) and the C-terminal cytosolic tail as compared to the intact molecule.
  • pXL3 and pXL4 were made to express the intact membrane-attached form of the RSV F molecule containing the TM and the cytosolic C-terminal tail.
  • the rationale for the presence of the intron II sequences in pXL2 and pXL4 was that this intron was reported to mediate the correct splicing of RNAs. Since mRNA for the RSV-F has been suspected to have a tendency towards aberrant splicing, the presence of the intron II sequences might help to overcome this. All four plasmid constructs were confirmed by DNA sequencing analysis.
  • Plasmid DNA was purified using plasmid mega kits from Qiagen (Chatsworth, Calif., USA) according to the manufacturer's instructions.
  • This Example describes the immunization of mice. Mice are susceptible to infection by RSV as described in ref. 16.
  • the anterior tibialis anterior muscles of groups of 9 BALB/c mice male, 6-8 week old (Jackson Lab., Bar Harbor, Me., USA) were bilaterally injected with 2 ⁇ 50 ⁇ g (1 ⁇ g/ ⁇ L in PBS) of pXL1-4, respectively.
  • the muscles were treated with 2 ⁇ 50 ⁇ L (10 ⁇ M in PBS) of cardiotoxin (Latoxan, France).
  • Pretreatment of the muscles with cardiotoxin has been reported to increase DNA uptake and to enhance the subsequent immune responses by the intramuscular route (ref. 24). These animals were similarly boosted a month later.
  • mice in the control group were immunized with a placebo plasmid containing identical vector backbone sequences without the RSV F gene according to the same schedule.
  • 100 ⁇ g of pXL2 (2 ⁇ g/ ⁇ L in PBS) were injected into the skin 1-2 cm distal from the tall base. The animals were similarly boosted a month later.
  • mice were challenged intranasally with 10 5 .4 plaque forming units (pfu) of mouse-adapted RSV, A2 subtype (obtained from Dr. P. Wyde, Baylor College of Medicine, Houston, Tex., USA). Lungs were aseptically removed 4 days later, weighed and homogenized in 2 mL of complete culture medium. The number of pfu in lung homogenates was determined in duplicates as previously described (ref. 19) using vaccine quality Vero cells. These data were subjected to statistic analysis using SigmaStat (Jandel Scientific Software, Guelph, Ont. Canada).
  • Sera obtained from immunized mice were analyzed for anti-RSV F antibody titres (IgG, IgG1 and IgG2a, respectively) by enzyme-linked immunosorbent assay (ELISA) and for RSV-specific plaque-reduction titres.
  • ELISA enzyme-linked immunosorbent assay
  • ELISA enzyme-linked immunosorbent assay
  • the secondary antibodies used were monospecific sheep anti-mouse IgG1 (Serotec, Toronto, Ont., Canada) and rat anti-mouse IgG2a (Zymed, San Francisco, Calif., USA) antibodies conjugated to alkaline phosphatase, respectively.
  • Plaque reduction titres were determined according to Prince et al (ref. 19) using vaccine quality Vero cells. Four-fold serial dilutions of immune sera were incubated with 50 pfu of RSV, Long strain (ATCC) in culture medium at 37° C. for 1 hr in the presence of 5% CO 2 . Vero cells were then infected with the mixture.
  • Plaques were fixed with 80% methanol and developed 5 days later using a mouse anti-RSV-F monoclonal IgG1 antibody and donkey antimouse IgG antibody conjugated to peroxidase (Jackson ImmunoRes., Mississauga, Ont. Canada).
  • the RSV-specific plaque reduction titre was defined as the dilution of serum sample yielding 60% reduction in the number of plaques.
  • Both ELISA and plaque reduction assays were performed in duplicates and data are expressed as the means of two determinations. These data were subjected to statistic analysis using SigmaStat (Jandel Scientific Software, Guelph, Ont. Canada).
  • spleens from 2 immunized mice were removed to prepare single cell suspensions which were pooled.
  • Splenocytes were incubated at 2.5 ⁇ 10 6 cells/mL in complete RPMI medium containing 10 U/mL murine interleukin 2 (IL-2) with ⁇ -irradiated (3,000 rads) syngeneic splenocytes (2.5 ⁇ 10 6 cells/mL) infected with 1 TCID 50 /cell RSV (Long strain) for 2 hr.
  • the source of murine IL-2 was supernatant of a mouse cell line constitutively secreting a high level of IL-2 obtained from Dr. H.
  • Target cells were 5 51 Cr-labelled uninfected BALB/c fibroblasts (BC cells) and persistently RSV-infected HCH14 fibroblasts, respectively. Washed responder cells were incubated with 2 ⁇ 10 3 target cells at varying effector to target ratios in 200 ⁇ L in 96-well V-bottomed tissue-culture plates for 4 hr at 37° C. Spontaneous and total chromium releases were determined by incubating target cells with either medium or 2.5% Triton-X 100 in the absence of responder lymphocytes.
  • Percentage specific chromium release was calculated as (counts-spontaneous counts)/(total counts-spontaneous counts) ⁇ 100. Tests were performed in triplicates and data are expressed as the means of three determinations. For antibody blocking studies in CTL assays, the effector cells were incubated for 1 hr with 10 ⁇ g/ml final of purified mAb to CD4 (GK1.5) (ref. 21) or mAb against murine CD8 (53-6.7) (ref. 22) before adding chromium labelled BC or BCH4 cells.
  • the chromium labelled target cells BC or BCH4 were incubated with 20 ⁇ L of culture supernate of hybridoma that secretes a mAb that recognizes K d and D d of class I MHC (34-1-2S) (ref. 23) prior to the addition of effector cells.
  • This Example describes the immunogenicity and protection by polynucleotide immunization by the intramuscular route.
  • This Example describes the influence of the route of administration of pXL2 on its immunogenicity and protective ability.
  • the i.m. and i.d. routes of DNA administration were compared for immunogenicity in terms of anti-RSV F antibody titres and RSV-specific plaque reduction titres. Analyses of the immune sera (Table 2 below) revealed that the i.d. route of DNA administration was as immunogenic as the i.m. route as judged by anti-RSV F IgG and IgG1 antibody responses as well as RSV-specific plaque reduction titres. However, only the i.m. route induced significant anti-RSV F IgG2a antibody responses, whereas the IgG2a isotype titre was negligible when the i.d. route was used. The i.m. and i.d.
  • This Example describes immunization studies in cotton rats using pXL2.
  • the RSV neutralizing titres on day +49 and +78 are shown in Tables 7(a) below and 7(b) below respectively.
  • Tables 7(a) The results shown in Table 7(a), on day +49 the animals immunized with live RSV and DNA immunization had substantial PSV serum neutralizing titres.
  • Boosting had no significant effect upon animals immunized with live RSV or by i.m. plasmid immunization.
  • RSV titres in nasal washes (upper respiratory tract) on day +82 are shown in Table 8 below.
  • RSV titres in the lungs (lower respiratory tract) on day +82 are shown in Table 9 below. All of the vaccines provided protection against lung infection but under these conditions, only live virus provided total protection against upper respiratory tract infection.
  • This Example describes the determination of local lung cytokine expression profile in mice imunized with pXL2 after RSV challenge.
  • RNA samples were prepared from lungs homogenized in TRIzol/ ⁇ -mercaptoethanol by chloroform extraction and isopropanol precipitation. Reverse transcriptase-polymerase chain reaction (RT-PCR) was then carried out on the RNA samples using either IL-4, IL-5 or IFN- ⁇ specific primers from Clone Tech.
  • the amplified products were then liquid-hybridized to cytokine-specific 32 P-labeled probes from Clone Tech, resolved on 5% polyacrylamide gels and quantitated by scanning of the radioactive signals in the gels.
  • Three mouse lungs were removed from each treatment group and analyzed for lung cytokine expression for a minimum of two times. The data is presented in FIG. 9 and represents the means and standard deviations of these determinations.
  • This Example describes the construction of a plasmid vector encoding the RSV F protein and containing the 5' UTR and signal peptide of Herpes Simplex Virus I (HSV I)gD.
  • Plasmid p82M35B was prepared following the scheme shown in FIG. 10. Plasmid pVR1012 (Vical) (FIG. 11; SEQ ID No: 6) containing the CMV promoter, intron A, and the BGH poly A sequences, was linearized with restriction enzyme Pst I and made blunt ended with T4 DNA polymerase. The rabbit ⁇ -globin intron II sequence was retrieved from plasmid pSG5 (Stratagene; ref. 14) by Cla I and Eco RI digestion, and the 0.6 kb fragment was isolated and made blunt ended by treatment with Klenow fragment polymerase. The rabbit ⁇ -globin intron II fragment was then ligated to the Pst I/blunt ended VR1012 plasmid (FIG. 10). This vector was then restricted with Eco RV and dephosphorylated.
  • the secreted form of RSV F was isolated from plasmid pXL2 (Example 1; FIG. 5) by digestion with Sal I, made blunt end by treatment with Klenow fragment polymerase, then restricted with Kpn I to produce a 5' Kpn I, 3'blunt ended fragment.
  • the HSV gD sequence was synthesized as a synthetic oligonucleotide having the DNA (SEQ ID No: 7) and derived amino acid (SEQ ID No: 8) sequence shown in FIG. 12.
  • the gD oligonucleotide has a 5' blunt end and 3' Kpn I recognition sequence.
  • a three-way ligation was performed with the isolated RSV F fragment, gD oligo and the VR1012 plasmid, to produce plasmid p82M35B (FIG. 10).
  • BHK cells were transfected with either p82M35B, its counterpart containing the autologous RSV F signal peptide (pXL2) or the vector backbone alone (placebo) using Lipofectin (Gtibco/BRL). Forty-eight hours post transfection, supernatant fractions were recovered and subjected to RSV F protein quantification using a F-specific enzyme-linked immunoabsorbent assay (ELISA). Three independent transfection assays were performed for each vector.
  • ELISA enzyme-linked immunoabsorbent assay
  • ELISAs were performed using one affinity-purified mouse monoclonal anti-RSV F antibody (2 ⁇ g/ml) as the capturing reagent and another biotinolated monoclonal anti-RSV F antibody (0.1 ⁇ g/ml) as the detection reagent.
  • Horseradish peroxidase-labelled avidin (Pierce) was subsequently used.
  • the RSV F standard protein used was purified from detergent-lysates of cultured virus by immunoaffinity chromatography.
  • Table 11 shows the results obtained. As seen in Table II, compared to placebo, both p82M35B and pXL2 mediated significant F protein expression/secretion from the BHK cells 48 hours post transfection. Furthermore, a markedly higher level of the F protein was consistently detected in the supernatant fraction of p82M35B-transfected BHK cells than that of pXL2-transfected cells, representing a 5.4-fold improvement over the latter.
  • Tibialis anterior muscles of BALB/c mice male, 6 to 8 weeks old (Jackson Lab., Bar Harbor, Me., USA) were bilaterally injected with 2 ⁇ 50 ⁇ g (1 ⁇ g/ ⁇ L in PBS) of p82M35B, pXL2 or the vector backbone alone (placebo).
  • the muscles were treated with 2 ⁇ 50 ⁇ L (10 ⁇ M in PBS) of cardiotoxin (Latoxan, France) to increase DNA uptake and enhance immune responses as reported by Davis et al., (ref. 24).
  • the animals were boosted with the same dose of plasmid DNA 6 weeks later.
  • mice in the positive control group were immunized intranasally (i.n.) with 10 6 plaque forming units (pfu) of a clinical RSV strain of the A2 subtype grown in Hep2 cells kindly provided by Dr. B. Graham (ref. 16).
  • Antisera obtained from immunized mice were analyzed for anti-RSV F IgG antibody titres using specific ELISA and for RSV-specific plaque-reduction titres.
  • ELISAs were performed using 96-well plates coated with immunoaffinity-purified RSV F protein (50 ng/mL) and 2-fold serial dilutions of immune sera.
  • a goat anti-mouse IgG antibody conjugated to alkaline phosphatase Jackson ImmunoRes., Mississauga, Ont., Canada
  • Plaque reduction titres were determined according to Prince et al. (ref. 19) using vaccine-quality Vero cells.
  • the present invention provides certain novel vectors containing genes encoding an RSV F proteins, methods of immunization using such vectors and methods of diagnosis using such vectors. Modifications are possible within the scope of this invention.
US09/262,927 1995-06-07 1999-03-05 Nucleic acid respiratory syncytial virus vaccines Expired - Lifetime US6083925A (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
US09/262,927 US6083925A (en) 1995-06-07 1999-03-05 Nucleic acid respiratory syncytial virus vaccines
CA002363593A CA2363593A1 (en) 1999-03-05 2000-03-03 Nucleic acid respiratory syncytial virus vaccines
MXPA01008966A MXPA01008966A (es) 1999-03-05 2000-03-03 Vacunas contra el virus sincicial respiratorio a base de acido nucleico.
AU29002/00A AU778144B2 (en) 1999-03-05 2000-03-03 Nucleic acid respiratory syncytial virus vaccines
PCT/CA2000/000227 WO2000053767A2 (en) 1999-03-05 2000-03-03 Nucleic acid respiratory syncytial virus vaccines
JP2000603388A JP3602448B2 (ja) 1999-03-05 2000-03-03 核酸呼吸器シンシチウムウイルスワクチン
NZ514528A NZ514528A (en) 1999-03-05 2000-03-03 Vectors comprising the sequnece for a respiratory syncytial virus (RSV) F protein with a heterologous signal sequence (HSV I gD) for vaccines
US09/570,383 US6486135B1 (en) 1995-06-07 2000-05-12 Nucleic acid respiratory syncytial virus vaccines

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US08/476,397 US6019980A (en) 1995-06-07 1995-06-07 Nucleic acid respiratory syncytial virus vaccines
US08/659,939 US5843913A (en) 1995-06-07 1996-06-07 Nucleic acid respiratory syncytial virus vaccines
US08/896,500 US6017897A (en) 1995-06-07 1997-07-18 Nucleic acid respiratory syncytial virus vaccines
US09/262,927 US6083925A (en) 1995-06-07 1999-03-05 Nucleic acid respiratory syncytial virus vaccines

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US08/896,500 Continuation-In-Part US6017897A (en) 1995-06-07 1997-07-18 Nucleic acid respiratory syncytial virus vaccines

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US09/570,383 Continuation US6486135B1 (en) 1995-06-07 2000-05-12 Nucleic acid respiratory syncytial virus vaccines

Publications (1)

Publication Number Publication Date
US6083925A true US6083925A (en) 2000-07-04

Family

ID=22999673

Family Applications (2)

Application Number Title Priority Date Filing Date
US09/262,927 Expired - Lifetime US6083925A (en) 1995-06-07 1999-03-05 Nucleic acid respiratory syncytial virus vaccines
US09/570,383 Expired - Fee Related US6486135B1 (en) 1995-06-07 2000-05-12 Nucleic acid respiratory syncytial virus vaccines

Family Applications After (1)

Application Number Title Priority Date Filing Date
US09/570,383 Expired - Fee Related US6486135B1 (en) 1995-06-07 2000-05-12 Nucleic acid respiratory syncytial virus vaccines

Country Status (7)

Country Link
US (2) US6083925A (ja)
JP (1) JP3602448B2 (ja)
AU (1) AU778144B2 (ja)
CA (1) CA2363593A1 (ja)
MX (1) MXPA01008966A (ja)
NZ (1) NZ514528A (ja)
WO (1) WO2000053767A2 (ja)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001096608A1 (en) * 2000-06-15 2001-12-20 Digene Corporation Detection of nucleic acids by type-specific hybrid capture method
US20070154884A1 (en) * 1997-12-12 2007-07-05 Lorincz Attila T Assessment of human papilloma virus-related disease
US7601497B2 (en) 2000-06-15 2009-10-13 Qiagen Gaithersburg, Inc. Detection of nucleic acids by target-specific hybrid capture method
US20090298187A1 (en) * 2008-04-17 2009-12-03 Qiagen Gaithersburg, Inc. Compositions, methods, and kits using synthetic probes for determining the presence of a target nucleic acid
US20100105060A1 (en) * 2008-10-27 2010-04-29 Qiagen Gaithersburg Inc. Fast results hybrid capture assay on an automated platform
US20100111989A1 (en) * 2006-12-21 2010-05-06 Pevion Biotech Ag Rsv f-protein and its use
US20100216147A1 (en) * 2009-01-28 2010-08-26 Qiagen Gaithersburg, Inc. Sequence-specific large volume sample preparation method and assay
US20110091496A1 (en) * 2008-01-19 2011-04-21 Graham Barney S Methods and compositions for the delivery of vaccines to disrupted epithelium
US20110217705A1 (en) * 2010-01-29 2011-09-08 Qiagen Gaithersburg Inc. Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
US9410146B2 (en) 2009-09-14 2016-08-09 Qiagen Gaithersburg Inc. Compositions and methods for recovery of nucleic acids or proteins from tissue samples fixed in cytology media
US9422593B2 (en) 2010-05-19 2016-08-23 Qiagen Gaithresburg, Inc Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
US9605303B2 (en) 2010-01-29 2017-03-28 Qiagen Gaithersburg, Inc. Method of determining and confirming the presence of an HPV in a sample
US9797000B2 (en) 2009-05-01 2017-10-24 Qiagen Gaithersburg Inc. Non-target amplification method for detection of RNA splice-forms in a sample
US9885092B2 (en) 2011-02-24 2018-02-06 Qiagen Gaithersburg Inc. Materials and methods for detection of HPV nucleic acids

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10221836A1 (de) * 2002-05-16 2003-12-04 Lohmann Animal Health Gmbh Attenuierung von Metapneumovirus
BRPI0908936A2 (pt) * 2008-03-14 2017-03-28 Sanofi Pasteur Biologics Co vacinas de flavivírus de replicação defeituosa e vetores de vacina
EP2367844A4 (en) * 2008-11-18 2012-08-01 Ligocyte Pharmaceuticals Inc RSV-F VLP AND MANUFACTURING METHOD AND METHOD OF USE THEREOF
US20120128713A1 (en) * 2009-03-16 2012-05-24 Sanofi Pasteur Limited Replication-Defective Flavivirus Vaccine Vectors Against Respiratory Syncytial Virus
KR101425404B1 (ko) * 2009-07-17 2014-08-01 한림대학교 산학협력단 리포좀에 포집된 올리고뉴클레오타이드 및 에피토프를 포함하는 면역증강용 조성물
EA201891945A3 (ru) 2012-08-01 2019-05-31 Бавариан Нордик А/С Вакцина рекомбинантного модифицированного вируса осповакцины анкара (mva) респираторно-синцитиального вируса (rsv)
US10368939B2 (en) 2015-10-29 2019-08-06 Covidien Lp Non-stick coated electrosurgical instruments and method for manufacturing the same
US10441349B2 (en) 2015-10-29 2019-10-15 Covidien Lp Non-stick coated electrosurgical instruments and method for manufacturing the same
US10709497B2 (en) 2017-09-22 2020-07-14 Covidien Lp Electrosurgical tissue sealing device with non-stick coating
US10973569B2 (en) 2017-09-22 2021-04-13 Covidien Lp Electrosurgical tissue sealing device with non-stick coating
CN109468323B (zh) * 2018-11-21 2022-03-11 新乡医学院 人工合成的内含子、哺乳动物细胞重组表达载体、哺乳动物宿主细胞、表达方法及其应用
US11207124B2 (en) 2019-07-08 2021-12-28 Covidien Lp Electrosurgical system for use with non-stick coated electrodes
US11369427B2 (en) 2019-12-17 2022-06-28 Covidien Lp System and method of manufacturing non-stick coated electrodes

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011092A1 (en) * 1989-03-21 1990-10-04 Vical, Inc. Expression of exogenous polynucleotide sequences in a vertebrate
WO1992007940A2 (en) * 1990-11-05 1992-05-14 Siba Kumar Samal Bovine respiratory syncytial virus genes
US5149650A (en) * 1986-01-14 1992-09-22 University Of North Carolina At Chapel Hill Vaccines for human respiratory virus
WO1993021310A1 (en) * 1992-04-21 1993-10-28 American Home Products Corporation Attenuated respiratory syncytial virus vaccine compositions
WO1994021797A1 (en) * 1993-03-18 1994-09-29 Merck & Co., Inc. Nucleic acid pharmaceuticals
US5589466A (en) * 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6019980A (en) * 1995-06-07 2000-02-01 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
CN1174099C (zh) * 1997-07-18 2004-11-03 康诺特实验室有限公司 编码呼吸道合胞病毒的g蛋白的核酸疫苗

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5149650A (en) * 1986-01-14 1992-09-22 University Of North Carolina At Chapel Hill Vaccines for human respiratory virus
WO1990011092A1 (en) * 1989-03-21 1990-10-04 Vical, Inc. Expression of exogenous polynucleotide sequences in a vertebrate
US5589466A (en) * 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
WO1992007940A2 (en) * 1990-11-05 1992-05-14 Siba Kumar Samal Bovine respiratory syncytial virus genes
WO1993021310A1 (en) * 1992-04-21 1993-10-28 American Home Products Corporation Attenuated respiratory syncytial virus vaccine compositions
WO1994021797A1 (en) * 1993-03-18 1994-09-29 Merck & Co., Inc. Nucleic acid pharmaceuticals

Non-Patent Citations (67)

* Cited by examiner, † Cited by third party
Title
Breathnack, R. et al., Nucl. Acids Res. 1983, 11:7119 7136. *
Breathnack, R. et al., Nucl. Acids Res. 1983, 11:7119-7136.
Chanock, Robert M. et al, Pediatrics vol. 90 No. 1, July 1992, pp 137 142. *
Chanock, Robert M. et al, Pediatrics vol. 90 No. 1, July 1992, pp 137-142.
Chapman, B.S. et al, Nucl. Acids Res. 1991, 19: 3979 3986. *
Chapman, B.S. et al, Nucl. Acids Res. 1991, 19: 3979-3986.
Collis et al (1990) Embo J. 9:233 240. *
Collis et al (1990) Embo J. 9:233-240.
Crowe, J.E., Vaccine 1995, 13:415 421. *
Crowe, J.E., Vaccine 1995, 13:415-421.
Davis et al, Vaccine 1994, 12: 1503 1509. *
Davis et al, Vaccine 1994, 12: 1503-1509.
Du, R.P. et al 1994, Biotechnology 12: 813 818. *
Du, R.P. et al 1994, Biotechnology 12: 813-818.
Furth et al. Analytical Biochemistry, 1992, 205: 365 368. *
Furth et al. Analytical Biochemistry, 1992, 205: 365-368.
Graham, B.S. et al., J. Mod. Virol. 1988 26: 153 162. *
Graham, B.S. et al., J. Mod. Virol. 1988 26: 153-162.
Green, S. et al., Nucl. Acids Res. 1988, 16:369. *
Groothuis et al, N. Engl. J. Med. 329:1524 1530, (1993). *
Groothuis et al, N. Engl. J. Med. 329:1524-1530, (1993).
Hemming et al, J. Infect. Dis., 152:1083 1087 (1985). *
Hemming et al, J. Infect. Dis., 152:1083-1087 (1985).
Johnson et al., J. Virol. 1987, 61: 3163 3165. *
Johnson et al., J. Virol. 1987, 61: 3163-3165.
Karasuyama & Melchers, Eur. J. Immunol. 18, 97 104, 1988. *
Karasuyama & Melchers, Eur. J. Immunol. 18, 97-104, 1988.
Katz, S.L.New Vaccine Development establishing priorities. vol. 1. Washington: National Academic Press: 1985 397 409. *
Katz, S.L.New Vaccine Development establishing priorities. vol. 1. Washington: National Academic Press: 1985 397-409.
Ledbetter, J.A. et al 1980, J. Exp. Med. 152:280 295. *
Ledbetter, J.A. et al 1980, J. Exp. Med. 152:280-295.
Lounsbach et al, Journal of General Virology 74, 2559 2565 (1993). *
Lounsbach et al, Journal of General Virology 74, 2559-2565 (1993).
McIntosh, K. Et al In: Fields BN, Knipe, DM, editors. Virology, New York: Raven Press 1990:1045 1072. *
McIntosh, K. Et al In: Fields BN, Knipe, DM, editors. Virology, New York: Raven Press 1990:1045-1072.
Nabel, G.J. et al Proc. Natl. Acad. Sci. USA 90:11307 11311 (1993). *
Nabel, G.J. et al Proc. Natl. Acad. Sci. USA 90:11307-11311 (1993).
Ozato Keiko et al, 1982, Transplantation 34:113 118. *
Ozato Keiko et al, 1982, Transplantation 34:113-118.
Paradiso et al, Pediatr. Infect. Dis. J. 13:792 798, (1994). *
Paradiso et al, Pediatr. Infect. Dis. J. 13:792-798, (1994).
Pemberton et al., J. Gen. Virol. 1987, 68:2177 2182. *
Pemberton et al., J. Gen. Virol. 1987, 68:2177-2182.
Pizzorno et al., J. Virol. Acids Res. 1988, G2:1167 1179. *
Pizzorno et al., J. Virol. Acids Res. 1988, G2:1167-1179.
Prince et al, J. Virol. 55:517;1985 Virus Res. 3; 193 206. *
Prince et al, J. Virol. 55:517;1985 Virus Res. 3; 193-206.
Prince et al, J. Virol., 61:1851 1854. (1987). *
Prince et al, J. Virol., 61:1851-1854. (1987).
Prince, G.A. et al, 1978. Ame J. Pathol. 93: 771 790. *
Prince, G.A. et al, 1978. Ame J. Pathol. 93: 771-790.
Tang et al (1993) J. Biol. Chem. 268:9522 9525. *
Tang et al (1993) J. Biol. Chem. 268:9522-9525.
Tang et al., Nature 1992, 356: 152 154. *
Tang et al., Nature 1992, 356: 152-154.
Ulmer, Current Opinion, Invest Drugs, 1993, 2:983 989. *
Ulmer, Current Opinion, Invest Drugs, 1993, 2:983-989.
Walsh et al, J. Infec. Dis., 155: 1198 1204, (1987) *
Walsh et al, J. Infec. Dis., 155: 1198-1204, (1987)
Wathen et al, J. Infect. Dis. 159: 255 264 (1989). *
Wathen et al, J. Infect. Dis. 159: 255-264 (1989).
Wertz et al, J. Virol 61: 293 301 (1987). *
Wertz et al, J. Virol 61: 293-301 (1987).
Wertz, G.W. et al, Biotechnology 1992, 20:151 176. *
Wertz, G.W. et al, Biotechnology 1992, 20:151-176.
Wilde, David B. et al., 1983, J. Immunol. 131:2178 2183. *
Wilde, David B. et al., 1983, J. Immunol. 131:2178-2183.

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070154884A1 (en) * 1997-12-12 2007-07-05 Lorincz Attila T Assessment of human papilloma virus-related disease
US20110097708A1 (en) * 1997-12-12 2011-04-28 Qiagen Gaithersburg Inc. Assessment of human papilloma virus-related disease
US7879546B2 (en) 1997-12-12 2011-02-01 Qiagen Gaithersburg Inc. Assessment of human papilloma virus-related disease
US7829691B2 (en) 2000-06-15 2010-11-09 Qiagen Gaithersburg, Inc. Detection of nucleic acids by type specific hybrid capture method
US20090162851A1 (en) * 2000-06-15 2009-06-25 Digene Corporation Detection of nucleic acids by type specific hybrid capture method
US7601497B2 (en) 2000-06-15 2009-10-13 Qiagen Gaithersburg, Inc. Detection of nucleic acids by target-specific hybrid capture method
US7645571B2 (en) 2000-06-15 2010-01-12 Qiagen Gaithersburg, Inc. Detection of nucleic acids by type-specific hybrid capture method
US8557973B2 (en) 2000-06-15 2013-10-15 Qiagen Gaithersburg, Inc. Detection of nucleic acids by target-specific hybrid capture method
WO2001096608A1 (en) * 2000-06-15 2001-12-20 Digene Corporation Detection of nucleic acids by type-specific hybrid capture method
US8389219B2 (en) 2000-06-15 2013-03-05 Qiagen Gaithersburg, Inc. Detection of nucleic acids by type-specific hybrid capture method
US20100036104A1 (en) * 2004-10-20 2010-02-11 Qiagen Gaithersburg Inc. Detection of nucleic acids by target-specific hybrid capture method
US9115410B2 (en) 2004-10-20 2015-08-25 Qiagen Gaithersburg, Inc. Detection of nucleic acids by target-specific hybrid capture method
US8901287B2 (en) 2004-10-20 2014-12-02 Qiagen Gaithersburg, Inc. Detection of nucleic acids by target-specific hybrid capture method
US20100111989A1 (en) * 2006-12-21 2010-05-06 Pevion Biotech Ag Rsv f-protein and its use
US8372963B2 (en) 2006-12-21 2013-02-12 Pevion Biotech Ag RSV F-protein and its use
US20110091496A1 (en) * 2008-01-19 2011-04-21 Graham Barney S Methods and compositions for the delivery of vaccines to disrupted epithelium
US20090298187A1 (en) * 2008-04-17 2009-12-03 Qiagen Gaithersburg, Inc. Compositions, methods, and kits using synthetic probes for determining the presence of a target nucleic acid
US20100105060A1 (en) * 2008-10-27 2010-04-29 Qiagen Gaithersburg Inc. Fast results hybrid capture assay on an automated platform
US8288520B2 (en) 2008-10-27 2012-10-16 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay and system
US8735564B2 (en) 2008-10-27 2014-05-27 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay and system
US8877436B2 (en) 2008-10-27 2014-11-04 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay on an automated platform
US20100216147A1 (en) * 2009-01-28 2010-08-26 Qiagen Gaithersburg, Inc. Sequence-specific large volume sample preparation method and assay
US9797000B2 (en) 2009-05-01 2017-10-24 Qiagen Gaithersburg Inc. Non-target amplification method for detection of RNA splice-forms in a sample
US9410146B2 (en) 2009-09-14 2016-08-09 Qiagen Gaithersburg Inc. Compositions and methods for recovery of nucleic acids or proteins from tissue samples fixed in cytology media
US20110217705A1 (en) * 2010-01-29 2011-09-08 Qiagen Gaithersburg Inc. Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
US9605303B2 (en) 2010-01-29 2017-03-28 Qiagen Gaithersburg, Inc. Method of determining and confirming the presence of an HPV in a sample
US9689047B2 (en) 2010-01-29 2017-06-27 Qiagen Gaithersburg Inc. Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
US9422593B2 (en) 2010-05-19 2016-08-23 Qiagen Gaithresburg, Inc Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
US9885092B2 (en) 2011-02-24 2018-02-06 Qiagen Gaithersburg Inc. Materials and methods for detection of HPV nucleic acids

Also Published As

Publication number Publication date
AU2900200A (en) 2000-09-28
MXPA01008966A (es) 2004-08-12
AU778144B2 (en) 2004-11-18
JP2002538788A (ja) 2002-11-19
WO2000053767A3 (en) 2000-12-28
WO2000053767A2 (en) 2000-09-14
JP3602448B2 (ja) 2004-12-15
NZ514528A (en) 2004-03-26
CA2363593A1 (en) 2000-09-14
US6486135B1 (en) 2002-11-26

Similar Documents

Publication Publication Date Title
US6022864A (en) Nucleic acid respiratory syncytial virus vaccines
US6083925A (en) Nucleic acid respiratory syncytial virus vaccines
AU667502B2 (en) Chimeric immunogens
CA2301891C (en) Rna respiratory synctial virus vaccines
AU756222B2 (en) Nucleic acid vaccines encoding G protein of respiratory syncytial virus
AU749345B2 (en) Alphavirus vectors for paramyxovirus vaccines
US6017897A (en) Nucleic acid respiratory syncytial virus vaccines
CN110996998A (zh) 表达rsv g的重组嵌合牛/人副流感病毒3及其用途
MXPA00000636A (en) Nucleic acid vaccines encoding g protein of respiratory syncytial virus
EP1707633A1 (en) Alphavirus vectors for paramyxovirus vaccines
MXPA00002236A (en) Rna respiratory syncytial virus vaccines

Legal Events

Date Code Title Description
AS Assignment

Owner name: CONNAUGHT LABORATORIES LIMITED, CANADA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LI, XIAOMAO;EWASYSHYN, MARY E.;SAMBHARA, SURYAPRAKASH;AND OTHERS;REEL/FRAME:010643/0303;SIGNING DATES FROM 19990317 TO 19990414

STCF Information on status: patent grant

Free format text: PATENTED CASE

FPAY Fee payment

Year of fee payment: 4

FPAY Fee payment

Year of fee payment: 8

FPAY Fee payment

Year of fee payment: 12